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An affinity for RNA

"DNA makes RNA makes protein," stated Francis Crick in 1958, outlining the pipeline by which information in genes determines what happens in cells and our bodies. Cells must produce specific proteins at precise times, a process which culminates in the translation of messenger RNAs (mRNAs) into proteins. This only happens if mRNAs survive long enough for delivery to specific locations in the cell and attachment to ribosomes during translation, the process by which proteins are synthesized. RNA-binding proteins (RBPs) carry out most of this work, but scientists have lacked a complete list of RBPs and their functions. Now many of the gaps are being filled in thanks to a new study from the lab of Markus Landthaler, collaborating with Matthias Selbach and Christoph Dieterich of the MDC and the group of Richard Bonneau at New York University. The work raises the total number of known RBPs from 600 to 800 and represents a crucial step in understanding the roles of the molecules in health and disease. The study appears in the June 8th edition of Molecular Cell.

This image shows positions in an untranslated region of messenger RNAs that are occupied by RNA-binding proteins and the extent to which they are evolutionarily conserved in mammalian species. Studying these positions revealed to the scientists that key positions on the RNAs have stayed constant over long stretches of evolutionary time, which almost always means that they have crucial functions in the lives of cells and organisms.

Many RBPs have been discovered in experiments that extract RNAs from cells and identify the proteins attached to them. But some molecules are lost during the extraction; others are nearly impossible to capture because they bind only very briefly to RNAs. Markus and his colleagues solved this problem by developing a method that cements RBPs firmly to RNAs, ensuring that even the most transient complexes remain intact.

The scientists achieved this by exchanging some of the building blocks of RNAs – nucleotides – with ultraviolet (UV) light-sensitive versions of the molecules. This has two effects. First, exposing cells to UV light triggers a process of crosslinking in which RBPs bind firmly to RNA. Additionally, when the molecules are extracted, scientists can see precisely where they dock onto the long, string-like RNAs. This can yield important new insights into the mechanisms that permit the molecules to bind; it also provides information about their functions.

This second part of the study is important partly because it can reveal how mutations in either RBPs or RNAs affect the binding process. Even slight alterations can change the outcome of these interactions and lead to disease.

The current project focused on human kidney cells, and each type of cell probably produces a few unique RBPs. Yet many are shared, and the close-up view of their binding sites should lead to the discovery of even more.

"Proteins and RNAs are generally large, complex molecules that bind at specific sites," Markus says. "Even if we know that two molecules interact, we usually lack a precise view of their points of contact. The project gives us a wealth of insights into the features that permit their interactions."

That information is crucial to a second method of finding RBPs, in which computers scan protein sequences for RNA-binding motifs. But such searches are based on amino acid sequence patterns from known RBPs, and Markus says that existing algorithms would have failed to predict at least 15 percent of the molecules detected in the study. A closer examination of the molecules' binding sites should reveal new motifs that can be used to rescan the catalogue of human proteins.

Uncovering binding sites in the proteins requires a complicated, molecule-by-molecule approach. In the paper, the researchers carried out this procedure for five proteins and are currently studying more.

But on the RNA side, things are clearer: the new method used in the paper precisely reveals docking sites on the RNA, allowing the scientists to draw a global map of the regulatory sequences bound by RBPs. Many are located in a long "tail" region of RNAs which is not directly translated into protein, but usually helps determine when, where, and if this occurs – through the activity of RBPs.

"Comparing the sites across animal species shows that these binding sites have experienced a low number of mutations over long stretches of evolutionary time," Markus says. "This is usually a sign that the regions are indeed functional."

It also means that disruptions in RBP-RNA interactions can have severe effects on human health. If an RBP called FXS is missing or defective in an embryo, the result is usually a condition called Fragile X Syndrome: a form of mental retardation.

Among the newly identified RBPs alone, fifteen proteins had already been associated with diseases. But researchers hadn't linked the symptoms to interactions between proteins and RNAs. The new information gives scientists a new focal point as they seek ways to treat the diseases.

Further experiments will be required to determine the exact functions of each RBP – in particular, how it regulates the stability and/or translation of an mRNA molecule. Markus also hopes that the information will help reveal how small genetic differences between individuals contribute to their overall health.

"The regulation of protein synthesis can affect health just as much as mutations or other changes in genes," Markus says. "Another finding of study supports the idea that RBPs act in a 'combinatorial' way – several of the molecules have to bind to an RNA to ensure its proper translation. Normal variations in the genes that we inherit cause subtle differences in the way these processes occur in different people. Although these differences can have significant effects over a lifetime, they are extremely difficult to define. The first step in that process is to build a much more extensive catalogue of RBPs. This puts us in a position to explore their functions – singly and in combinations."